One of the most intriguing questions about electron transfer proteins is how the protein modifies the electron transfer properties of a given type of redox site. Knowledge of the structural origins of their electron transfer properties is crucial in understanding the molecular basis of a variety of metabolic processes, diseases involving these processes, and drug design targeting these processes. The overall goal of this research is to understand the properties of electron transfer proteins at a molecular level. The focus is on the iron-sulfur proteins, which are ubiquitous proteins involved in a variety of fundamental processes such as respiration, photosynthesis, biosynthesis, and biodegradation. Our premise is that the structural origins of the reduction potentials for these proteins are mainly the electrostatic effects of the backbone, polar side chains, and solvent. Since polar contributions depend on both orientation and distance and there are many such small contributions, they are almost impossible sort out by structural or mutational data alone. Our research has led to the development of a strategy for identifying these contributions. This strategy will now be applied to other proteins and the influence of these contributions on activation energies will be examined. Our premise further is that the electronic structure of the redox site is environment independent. This will be used to determine the effects of metal and ligand substitution and cluster conversion. All of these results will then be used to define structure/function relationships. Our approach uses electrostatic potential calculations of experimental structures, molecular dynamics simulation methods, electronic structure methods, and sequence analysis. Studies will be of the [1Fe] rubredoxins, the [2Fe-2S] Rieske proteins, and the 2[4Fe-4S] ferredoxins. The specific aims are as follows. Aim 1. Origins of differences in E due to changes in the outer sphere will be determined. Our hypothesis is that sequence differences can affect E by altering the electrostatic contributions of protein polar groups and solvent. Aim 2. Origins of differences in E due to changes in the inner sphere will be determined. Our hypothesis is that the electronic contribution is approximately independent of the protein environment. Aim 3. Effects of sequence on electron transfer between sites will be determined. Our hypothesis is that sequence differences can affect the reorganization energy for electron transfer. Aim 4. Co evolution of E sequence determinants, fold, and function will be determined. Our hypothesis is that sequence determinants of E should be conserved within functionally related proteins.